As used herein, the term "angiogenesis" means the generation of new blood vessels into a tissue or organ. Under normal physiological conditions, humans or animals undergo angiogenesis only in very specific restricted situations. For example, angiogenesis is normally observed in wound healing, the female reproductive cycle and embryonic development..sup.1 Angiogenesis is also critical in the progression of many disease states, such as hemangioma, endometriosis, solid tumors and macular degeneration.
Through angiogenesis, endothelial cells and pericytes, surrounded by a basement membrane, form capillary blood vessels. Angiogenesis begins with the erosion of the basement membrane by enzymes released by endothelial cells and leukocytes. The endothelial cells, which line the lumen of blood vessels, then protrude through the basement membrane. Angiogenic stimulants induce the endothelial cells to migrate through the eroded basement membrane. The migrating cells form a "sprout" off the parent blood vessel, where the endothelial cells undergo mitosis and proliferate. The endothelial sprouts merge with each other to form capillary loops, creating the new blood vessel.
Angiogenesis occurs during the cyclical changes of the female reproductive system. In preparation for fertilization, the theca interna of the ovaries becomes richly vascularized to support the growth of a follicle. During ovulation, the follicle ruptures and an egg is released to the uterus. If fertilization occurs, the ruptured follicle then is invaded by thecal vessels and forms the corpus luteum. The corpus luteum releases hormones that regulate reproductive processes, including the thickening of the endometrium for the implantation of the fertilized ovum. After the blastocyst implants in the endometrium, the placenta develops and provides nutrients for the growing embryo. Throughout all of these stages of mammalian reproduction, angiogenesis is critical to further development.
Specifically, angiogenesis occurs during follicular growth, formation of the corpus luteum, growth of the endometrium, and development of the placenta and embryo after conception..sup.1,2,3,4 These angiogenic episodes are self-limiting and presumably tightly regulated. During the menstrual cycle, the spiral arteries undergo substantial anatomical changes. As the endometrium thickens three to five fold during the next menstrual cycle, the remnants of the spiral arteries must undergo substantial growth and give rise to a new capillary bed in order to maintain the integrity of the rapidly growing stroma. This uterine angiogenesis provides an existing vascular supply for the trophoblast to invade if fertilization of the ovum occurs. Following fertilization, the first stage of implantation is the adhesion of the blastocyst to the endometrial epithelium. This is followed by the penetration of the trophoblast through the epithelial lining..sup.7
Physiological changes in the ovary are especially critical for the proper functioning of the female reproductive cycle. In the ovary, during the course of follicular growth, the theca interna becomes richly vascularized. Follicular maturation is associated with increasing angiogenesis, whereas follicles undergoing atresia are associated with decreasing vascularity..sup.8 Following ovulation, growth factors are expressed which induce the thecal vessels to grow and invade the ruptured follicle and form a complex capillary network which nourishes the developing corpus luteum..sup.9,10,11,2,12 Approximately 50% of the cells of the mature bovine corpus luteum are endothelial cells, and in the primate corpus luteal endothelial cells comprise 85% of proliferating cells..sup.4
The mitotic activity of these endothelial cells is highest during the early luteal phase, persists but declines during the midluteal phase and is minimal during luteal regression. Corpus luteal maintenance during pregnancy is associated with continued angiogenesis..sup.4 This has been demonstrated in the pregnant rat where the labeling index of endothelial cells in the corpus luteum increases and peaks on embryonic day 14 (E14) which correlates with further growth of the corpus luteum..sup.5 Angiogenesis and its hormonal control in the corpus luteum of the pregnant rat..sup.13 Thus, the exponential growth of the corpus luteum is associated with aggressive neovascularization. This vasculature plays a vital role in providing nutrients and trophic factors to and transporting secreted hormones from the developing follicle and corpus luteum..sup.14
The factors controlling ovarian angiogenesis are just beginning to be fully understood. Two growth factors demonstrated in the ovary are basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). Cultured granulosa cells produce bFGF, the release of which may be modulated by heparin sulfate proteoglycans which are produced under gonadotropin modulation. Recent evidence suggests that vascular endothelial growth factor (VEGF), an endothelial cell specific mitogen and permeability factor, may play an integral role in ovarian angiogenesis..sup.15,16,17
VEGF, a secreted homodimeric glycoprotein, is under gonadotropin control. It is both temporally and spatially correlated with active angiogenesis in the theca and the corpus luteum. Thus, as is the case with other tissues throughout the body, the ovary has multiple stimulators of angiogenesis including.
VEGF and bFGF which are likely to be in balance with endogenous inhibitors to achieve the appropriate level of blood vessel growth..sup.18,19,20,21,22,23 In addition, the uterus undergoes dramatic physiological changes in the course of the female reproductive cycle. Under hormonal regulation by estrogen and progesterone, the uterus undergoes differentiation into a structure which is capable of supporting the implantation of a blastocyst. The cyclical maturation of the endometrium in the uterus is driven by the mitogenic activity of the spiral arteries. Following implantation, the maternal spiral arteries within the endometrium are invaded by trophoblasts that adhere to and migrate along the surface of the vascular endothelial cells.
Currently, biochemical female reproductive control is primarily accomplished through regimens of hormones such as estrogen and progesterone. Although this method of birth control is quite effective in preventing pregnancy, there are many side effects to the administration of additional hormones. Often such forms of birth control cause emotional and physiological disturbances, resulting in mood shifts or loss of libido. Health risks such as stroke and heart problems increase for women who smoke while using hormonal birth control. In addition, these forms of fertility control are only potent when taken on a regularly basis. The administration of hormones is advised to be discontinued should fertilization occur to diminish chances of health complications..sup.24
Many factors, chemical as well as mechanical, have been shown to be capable of promoting or inhibiting angiogenesis in vivo and in vitro..sup.25 Although the presence of angiogenesis promoting factors during growth and development of the ovaries, uterus, and placenta have been evaluated, the effects of angiogenesis inhibiting factors on these organs have not be elucidated. Since angiogenesis is an important component in the female reproductive system, it might be possible to regulate reproductive processes with angiogenesis inhibiting factors.
There are a large number of known angiogenesis inhibiting compounds which will undoubtedly continue to grow as scientific research continues. Some of the currently known angiogenesis inhibiting compounds are: AGM-1470 (TNP-470) or antagonists to one of its receptors MetAP-2; growth factor antagonists or antibodies to growth factors (including VEGF or bFGF); growth factor receptor antagonists or antibodies to growth factor receptors; inhibitors of metalloproteinases including TIMP, batimastat (BB-94), and marimastat; tyrosine kinase inhibitors including genistein and SU5416; integrin antagonists including antagonists alphaVbeta3/5 or antibodies to integrins; retinoids including retinoic acid or the synthetic retinoid fenretinide; steroids 11.alpha.-epihydrocortisol, corteloxone, tetrahydrocortisone and 17.alpha.-hydoxyprogesterone; protein kinase inhibitors including staurosporine and MDL 27032; vitamin D derivatives including 22-oxa-1 alpha, and 25-dihydroxyvitamin D3; arachidonic acid inhibitors including indomethacin and sulindac; tetracycline derivatives including minocycline; thalidomide derivatives; 2-methoxyestradiol; tumor necrosis factor-alpha; interferon-gamma-inducible protein 10 (IP-10); interleukin 1 and interleukin 12; interferon alpha, beta or gamma; angiostatin.TM. protein or plasminogen fragments; endostatin.TM. protein or collagen 18 fragments; proliferin-related protein; group B streptococcus toxin; CM101; CAI; troponin I; squalamine; nitric oxide synthase inhibitors including L-NAME; thrombospondin; wortmannin; amiloride; spironolactone; ursodeoxycholic acid; bufalin; suramin; tecogalan sodium; linoleic acid; captopril; irsogladine; FR-118487; triterpene acids; castanospermine; leukemia inhibitory factor; lavendustin A; platelet factor-4; herbimycin A; diaminoantraquinone; taxol; aurintricarboxylic acid; DS-4152; pentosan polysulphite; radicicol; fragments of human prolactin; erbstatin; eponemycin; shark cartilage; protamine; Louisianin A, C and D; PAF antagonist WEB 2086; auranofin; ascorbic ethers; and sulfated polysaccharide D 4152.
AGM-1470 (O-chloroacetylcarbamoyl fumagillol) is an analog of fumagillin with well-described angiogenesis inhibiting activities that is highly selective, potent, and non-toxic..sup.26 AGM-1470 inhibits endothelial cell proliferation at concentrations that are inactive for other cell types..sup.27 Fumagillin is a chemical produced by Aspergillus fumigatus which acts as a potent angiogenesis inhibitor in vitro and in vivo. AGM-1470 is 50 times more active and much less toxic than the parent compound. It potently inhibits endothelial proliferation and migration as well as angiogenesis in the chick chorioallantoic membrane. AGM-1470 has shown marked antineoplastic activity in animal models and is in clinical trials for the treatment of human neoplastic tumors and other angiogenic disorders.
Although shown to be an effective inhibitor of tumor angiogenesis, angiogenesis inhibiting compounds have not heretofore been shown to be effective in inhibiting angiogenesis in female reproductive and embryonic tissues. The angiogenesis of female reproductive tissues differs from angiogenesis in other tissues, such as in a tumor, as they are affected by different growth factors and under different regulatory mechanisms. Additionally, inhibition of angiogenesis in the female reproductive system has not heretofore been shown to disrupt or regulate critical events in the reproductive cycle. Thus, the successful use of angiogenesis inhibitors for contraception or treatment of disease in the tissues of the female reproductive system has not been previously demonstrated as possible, effective, safe, and without irreversible effects on the female reproductive system.
Another impediment to the use of angiogenesis inhibitors as contraceptives relates to the possible risk of inducing birth defects if contraception fails. This concern derives from the recent observation that thalidomide is an angiogenesis inhibitor..sup.28 Thalidomide produces unique birth defects when mothers ingest the drug during the first trimester. However, we believed that thalidomide had a unique capacity to induce limb defects by specific interaction with receptors in the limbs and through local concentration of the drug in the limbs. Therefore, we attempted herein to establish that other angiogenesis inhibitors can be used as contraceptives without inducing birth defects.
It is clear that angiogenesis plays a major role in the events encompassing reproduction. Proliferation of blood vessels is seen coincident with ovulation, corpus luteal growth, decidualization and placental formation. If this angiogenic activity could be reversibly repressed or eliminated, fertility and pathological reproductive disorders could be controlled. What is needed, therefore, is to demonstrate that angiogenesis inhibitors can inhibit the female reproductive system, without side-effects or irreversibility of the physiological changes.